Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 70 No. 6 pp. 1003ñ1009, 2013 ISSN 0001-6837Polish Pharmaceutical Society

The family Ephedraceae has the only genusEphedra L., which consists of about 50 species ofperennials and shrubs in the world. Ephedra L. gen-erally grows wild in arid and semiarid climates anddistributed mainly in the temperate zones of Europe,Asia and North America. They are called ìjoint-pineî, ìjoint firî, ìsea grapeî, ìmormon-teaî orìshrubby horsetailsî in English, and ìormakî, ìrishbozî or ìalijonakî in Persian (1). This genus is com-monly used by the Chinese people as a folk medicinefor treatment of allergies, bronchial asthma, chills,colds, coughs, edema, fever, flu, headaches, and nasalcongestion. The main chemical compounds havebeen identified and isolated from Ephedra extract isalkaloids group such as ephedrine, pseudoephedrine,and norpseudoephedrine (2-4). In flora of Iran, 12species of Ephedra has been reported (1). Ephedrapachyclada Boiss. (ìHoomî or ìHoomaî in Persian)

is distributed from Sinai to Pakistan, the Zagros rangein Iran and extending south to Yemen and Oman. Leeand Lee (5) reported that quinaldic acid isolated fromthe stems of E. pachyclada had antibacterial activityagainst Clostridium difficile and C. perfringens, whilehad no effect on the growth of Bifidobacteriumbifidum, Lactobacillus acidophilus and L. casei.

Gastric ulcer is a major gastrointestinal disor-der, which occurs due to an imbalance between theoffensive and defensive factors (6, 7). Various fac-tors, including stress, smoking, nutritional deficien-cies, Helicobacter pylori and ingestion of nonsteroidal anti-inflammatory drugs increase the gas-tric ulcer incidence (8, 9). Current chemical drugsfor treatment of peptic ulcer are associated withmany side effects, tolerance to drugs and occurrenceof relapse. These problems are the basis for devel-oping of new drugs with herbal origin (10). In folk

NATURAL DRUGS

HEALING EFFECT OF HYDRO-ALCOHOLIC EXTRACT OF EPHEDRAPACHYCLADA BOISS. IN EXPERIMENTAL GASTRIC ULCER IN RAT

ABDOLLAH GHASEMI PIRBALOUTI1*, MEHDI AMIRMOHAMMADI2 SHAHRZAD AZIZI3

and LYLE CRAKER4

1Shahrekord Branch, Islamic Azad University, Research Center for Medicinal Plants & Ethno-veterinary,P.O. Box: 166, Shahrekord, Iran

2Jiroft Branch, Islamic Azad University, Young Researchers Club, Jiroft, Kerman, Iran3Department of Pathobiology, Veterinary Medicine Faculty, Shahid Bahonar University of Kerman,

Kerman, Iran4Medicinal Plants Program, College of Natural Sciences, Massachusetts University,

Amherst, 01003, MA, USA

Abstract: Ephedra pachyclada Boiss. (family Ephedraceae) is a medicinal plant very frequently cited as actingagainst gastrointestinal disorders in ethno-pharmacological inventories of the Kerman region of Iran. This studywas done to evaluate the effect of hydro-alcoholic extract from the stems of E. pachyclada for treatment of gas-tric ulcers induced by ethanol in Wistar rats. Experimental treatments were the hydro-alcoholic extract of E.pachyclada (250, 500, and 1000 mg/kg, orally), omperazole as standard drug (20 mg/kg, orally), and controlgroup. Ulcer index in mm2 and histological examination were evaluated. On 3, 6, 9 and 12 day after treatments,the hydro-alcoholic extract of E. pachyclata (1000 mg/kg) produced 51, 72, 98.8 and 100% and omperazolealso produced 53, 79, 93 and 100% curative effect for gastric mucosal damage in ethanol model, respectively.The results of the histopalogical analysis indicated the hydro-alcoholic extract of E. pachyclada at 1000 mg/kgwas effective in experimentally healing rat ulcers. E. pachyclada accelerated ulcer healing in rats and, thus sup-ports its folk medicine use by Kerman people.

Keywords: Ephedra pachyclada, ulcer, hydro-alcoholic extract

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* Corresponding author: e-mail: ghasemi@iaushk.ac.ir; phone: 00983813361060, fax: 00983813361031

1004 ABDOLLAH GHASEMI PIRBALOUTI et al.

knowledge, medicinal plants are used for treatmentof various disorders with little information of theirpharmacological uses (11). For example, E. pachy-clada popularly known as ìKhimokî used for treat-ments gastric disorders by Kerman tribal, in CentralIran (12).

To our knowledge, no documented reports onthe effect of hydro-alcoholic extract of E. pachycla-da on gastric ulcers are available. Therefore, the aimof this study was to evaluate the protective effect ofE. pachyclada hydro-alcoholic extract against gas-tric ulcers induced by ethanol in rat.

MATERIALS AND METHODS

Plant extract preparation

The stems of E. pachyclada were collectedfrom Kerman, Iran in June 2011. The sample ofplant was identified by regional floras and voucherspecimen was deposited at the Herbarium ofAgriculture and Natural Resources Research Centerof Kerman province, Iran (No. 7667). The stems ofE. pachyclada were dried at room temperature(30OC) and then powdered. The hydro-alcoholicextract was obtained by maceration of the crudeplant powder with ethanol/water (70/30) for fourdays in a chamber temperature (35 ± 5OC). Thehydro-alcoholic extract was filtered using a sterilecloth sheet, then dried under reduced pressure attemperature below 45OC with rotary evaporator andgave a dark red residue with yield 7%. The extractsamples were stored in universal bottles and refrig-erated at 4OC prior to use.

Determination of total phenolic content

Total phenolic compound content in extractwas determined by FolinñCiocalteu method withsome modifications (13). In this method, 0.5 mL ofthe sample was mixed with 2.5 mL of Folin-Ciocalteu phenol reagent. After 5 min at 37OC, 2 mLof saturated Na2CO3 (7.5%) solution was added tothe mixture and it was made up to 10 mL by addingdeionized distilled water. The mixture was kept for120 min at room temperature in the dark. Theabsorbance was measured at 765 nm against thereagent blank with a Shimadzu UVñVis spectropho-tometer (Shimadzu Corp., Japan). Gallic acid(Sigma, G-7384) was used as the reference standard.The total phenolic content is expressed as mg of gal-lic acid equivalents per gram of extract on dry basis.

Experimental animals

Male Wistar rats (200-250 g) of three monthswere used. The animals were housed in standard

environmental conditions of temperature (22 ± 3OC),humidity (60 ± 5%) and a 12-h light/dark cycle.During experimental time, rats were given standardpellet diet (Pastor Institute, Iran) and water ad libi-tum. The rats were used for the experiment after oneweek of acclimatization period. All the procedureswere approved by the Medical Ethics Committee ofShahrekord University of Medical Sciences.

Ethanol induced ulcer

After fasting for 48 h and supplied withsucrose 8% during the fasting period, each animalreceived 1 mL of absolute ethanol (99.6%) orally(14). The experimental treatments were submitted tothe treatments with vehicle, omeprazole (20 mg/kg),hydro-alcoholic extract (250, 500, and 1000 mg/kg)1 h after induction of gastric injury by absoluteethanol.

Determination of ulcer index

On 3, 6, 9 and 14 days after induced ulcer,ulcer area were measured in mm2 by tracing thewound boundaries on a transparent paper. The ani-mals were sacrificed 1 h later using an overdose ofether, and the stomach removed and observed forulcers in the glandular and nonglandular region (15).The surface area of each lesion was measured andscored as described by Tan et al. (16). The ulcerindex for each rat was taken as the mean ulcer score.The percentage of curative ratio (%CR) was calcu-lated as described by Nguelefack et al. (17) using thefollowing formula:

% US(c) - US(t)% CR = óóóóóóó ◊ 100

US(c)

where US(c) = ulcer surface area in control andUS(t) = ulcer surface area in treated animals.

Histopathology analysis

On 3, 6, 9 and 14 day after induction, theexperiment was terminated and the ulcer area wasremoved from the surviving rats for histologicalanalysis. The samples were fixed in 10% formalde-hyde solution and were embedded in paraffin wax.A 5 µm thickness sections were stained with hema-toxylin and eosin and observed for the histopatho-logical changes under light microscope (OlympusBX51).

Statistical analysis

The results were expressed as the mean ± SD.The difference between the mean ulcer indexes ofexperimental groups were analyzed using ANOVAand followed by Tukey test; p ≤ 0.05 was considered

Healing effect of hydro-alcoholic extract of... 1005

statistically significant. All data processing was per-formed with SPSS software version 17.

RESULTS AND DISCUSSION

Ulcer index

The effect of treatments was investigated onexperimental gastric ulcers induced by ethanol inrats (Table 1) Statistically, the percentage of ulcerindex indicated that there were significant differ-ences between treatments on 3, 6, 9 and 14 daysafter induced ulcer (Table 1). The ulcer index incontrol group was highly significant (p < 0.01) incomparison with the other groups during the exper-imental course (Table 1). The results indicated thatomeperazole and the hydro-alcoholic extract at 500and 1000 mg/kg indicated 99.8, 100 and 100% heal-ing effect, respectively, after 14 days of experiment(Table 2).

Histopathology investigation

At 3rd day of treatment, all groups showedgrossly severe mucosal ulceration, necrosis and hem-orrhage. Ulcers in different forms and sizes were dis-

persed mainly in glandular and less in nonglandularpart of stomach. The mucosal stomach was hyper-emic and the margin ulcers were edematous andswollen. Hyperemia was more evident in the controlgroups than in the others. Histopathologically, necro-sis and slaughing of gastric mucosa were occurredand erosions and ulcers in different degrees affectedmucosal stomach. Lamina propria showed hyper-emia and hemorrhage. Inflammatory cells, especial-ly neutrophils and a few eosinophils, infiltratedaround the marginal ulcers. Submucosal layer wasedematous and thickened by a lot of fibrin exudatesand hemorrhage (Fig. 1). At 6th day, the hyperemiaand hemorrhage were decreased in all treated groups,especially in omeperazole treated group. Varioussizes of ulcers and erosions were observed yet andtheir surfaces were covered superficially by a fibri-nonecrotic layer. The control group showed severehemorrhage and hyperemia with extended ulcers.Granulation tissue was developing at the base ofulcerated areas. Lamina propria and submucosallayer showed moderate hyperemia and inflammatorycells were scattered in these layers. At 9th day,except of control group, mucosal hemorrhage and

Table 1. Effect of the treatments on ulcer index.

Ulcer index (mm2)

Treatments 3rd 6th 9th 14th

Control 321.66 ± 17.90Ü bá 251.00 ± 26.83 b 285.66 ± 7.85 c 314.66 ± 17.75 b

Standard drug 150.00 ± 33.20 ab 51.00 ± 11.84 a 19.66 ± 8.19 a 0.00 ±0.00 a (omperazole)

Extract 250 mg/kg 139.00 ± 43.31 a 104.66 ± 12.81 a 13.00 ± 3.05 a 6.00 ± 2.08 a

Extract 500 mg/kg 137.33 ± 36.33 a 75.33 ± 7.42 a 4.00 ±1.732 a 0.33 ± 0.33 a

Extract 1000 mg/kg 157.66 ± 56.29 b 70.00 ± 25.54 a 3.33 ± 1.20 a 0.00 ± 0.00 a

ANOVA p ≤ 0.05 p < 0.01 p < 0.01 p < 0.01

ÜEach value represents the mean ± S.D. n = 3. áMeans with different letter in a row are statistically significant at 5% level probability.

Table 2. Effect of the treatments on curative ratio (% CR).

Curative ratio (% CR)

Treatments 3th 6th 9th 14th

Control - - - -

Standard drug 98 93.1 79.6 53.3(omperazole)

Extract 250 mg/kg 99 95 58.3 54.7

Extract 500 mg/kg 100 98.5 70 57.3

Extract 1000 mg/kg 100 98.8 72.1 51

1006 ABDOLLAH GHASEMI PIRBALOUTI et al.

Figure 1. Gastric erosions, ulcers and hemorrhage induced byethanol in rat; a) rat treated with E. pachyclada extract (1000mg/kg), b) control group (saline), c) histopathologic features ofstomach show mucosal ulcers (arrows) and thickening of submu-cosal layer by fibrin accumulation and inflammatory cells (aster-isk) (magnification 40◊, hematoxylin + eosin)

Figure 2. Ethanol-induced ulcers in ratís stomach 9 days aftertreatment; a-c) small erosions (arrows) in rat treated with E.pachyclada extracts at 1000, and 250 mg/kg and omeperazole (20mg/kg), respectively (arrows). Treated group with the extract (250mg/kg) show more erosion and ulcer in the stomach (arrows), d)histopathologic features of stomach show superficial erosion inthe central part of healed ulcer (arrow) (magnification 100◊,hematoxylin + eosin)

Healing effect of hydro-alcoholic extract of... 1007

Figure 3. Ethanol-induced ulcers in ratís stomach 14 days after treatment; a-c) rat treated with E. pachyclada extracts at 1000, and 250mg/kg and omeprazole (20 mg/kg), respectively. Macroscopic small erosions and ulcers are visible in treated rat with 250 mg/kg extract(arrows), d) control group (saline), e) histopathologic features of stomach treated with E. pachyclada extract (1000 mg/kg) show repair ofulcerated area (arrow) (magnification 100◊, hematoxylin + eosin), f) histopathologic features of stomach in rat treated with E. pachycladaextract (250 mg/kg) show superficial erosion in the central part of healed ulcer (arrow) (magnification 100◊, hematoxylin + eosin)

1008 ABDOLLAH GHASEMI PIRBALOUTI et al.

hyperemia were decreased remarkably in the extractgroups (500 and 1000 mg/kg) and omeprazole group.The ulcerated lesions were healed but small erosionsremained. The extract group at 250 mg/kg, showedmore hemorrhage and hyperemia, and presence ofsome ulcers were noticeable. Thickness of gastricmucosa was variable in different areas and treatedrats had thin-walled stomachs. Microscopic analysisshowed well developed regeneration of epithelium inulcers margin and throughout the ulcerated areas.New formed gastric glands had simple columnarepithelium and their lumens were dilated. The super-ficial erosions were located in the central portion ofthe healed ulcers (Fig. 2). The base of healed ulcerwas formed by the dense connective tissue scar. Inthe submucosa, a great number of newly formed ves-sels were noticeable in the extracts treated groupsespecially at the higher doses. At 14th day, no gastricerosions or ulcers were found in the rats whichreceived the extract (1000 mg/kg) and omeperazolebut very small erosions were visible yet at dose 250and 500 mg/kg of extract. Control group showedsevere hemorrhage and mucosal damage (Fig. 3). Weobserved a thicker regenerative mucosa in treatedanimals for 14 days when compared to the controlgroup. The thickness of regenerated areas was notthe same in different parts of mucosa. In all groups,there were infiltration of a few lymphocytes andeosinophils in submucosal layer and the base ofhealed regions.

This study investigated the healing activity ofthe hydro-alcoholic extract of E. pachyclada on gas-tric ulcers induced by ethanol in rats. In experimen-tal studies, intragastrical ethanol was used as themost common ulcerogenic agent that creates severegastric hemorrhagic erosions and ulcers (18). Thepathogenesis of gastric lesions induced by ethanol ismultifactorial, including depletion of gastric wallmucus content (19), disrupting its barrier, stimulat-ing and changes of microvasculature in few minutesafter its application. Combination of rapid andsevere vasoconstriction and then following vasodi-lation induces damage to capillaries of mucosallayer (20, 21). There is in agreement that thedestructive effects of ethanol on gastric mucosa areresults of enhanced lipid peroxidation. The oxygenfree radicals cause lipid peroxidation and conse-quently gastric mucosal lesions that are induced bysome agents such as indomethacin, alcohol andaspirin in rats (22).

Liquorice and vitamin E in rats are reducedunder ethanol treatment and caused significantchanges in the ulcer index. The presence of thesephytochemicals may be responsible for the gastro-

protective action of the plant extract. Schmeda-Hirschmann and Yesilada (23) reported that chemi-cal compounds isolated from medicinal plants havevaried antiulcer activity. Our results indicated thatregenerative mucosa were thicker after 14 days withE. pachyclada hydro-alcoholic extract (500 and1000 mg/kg) in comparison with the extract at 250mg/kg and control group. Behravan et al. (24)reported that gastric ulcerogenicity of the extract ofAnacardium occidentale (cashew nut) at the doses of300, 400 and 800 mg/kg was lower than for the sim-ilar doses of indomethacin.

Results of our study indicated that total pheno-lic content in the extract of E. pachyclada was 45mg of GAE/g dry weight. It has been demonstratedthat many medicinal plants or herbs can contain awide variety of free radical scavenging molecules,such as phenolic compounds (phenolic acids,flavonoids, quinones, coumarins, lignans, stilbenes,tannins), nitrogen compounds (alkaloids, amines,betalains) etc. which are effective in healing experi-mentally induced gastric ulcers (25).

Finally, we can suggest that the hydro-alco-holic extract of E. pachyclada was effective inexperimentally healing rat ulcers. The hydro-alco-holic extract of E. pachyclada at 1000 mg/kg pro-duced a significant gastric ulcer when comparedwith the negative control and omeperazole (standarddrug). Further, the results of current study prove theanti-ulcer effect and suggest that it may be due toantioxidant mechanism of action.

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Received: 29. 03. 2012